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EJBRAT 7(3) 2017

Volume 7

Number 3

July-September 2017

European Journal

of Biological Research

MNiSW points 2016:

11

Index Copernicus 2015:

93.39

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European Journal of Biological Research, Volume 7, Issue 3, July-September 2017 European Journal of Biological Research

ISSN 2449-8955

Editor-in-Chief

Tomasz M. Karpiński

Poznań University of Medical Sciences, Poznań, Poland

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Institute of Natural Fibres and Medicinal Plants, Poznań, Poland

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Copyright: © The Author(s) 2017. European Journal of Biological Research © 2017 T.M.Karpiński. All articles and abstracts are open-access, distributed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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European Journal of Biological Research, Volume 7, Issue 3, July-September 2017

Contents

154-164

165-171

172-190

191-201

202-206

207-222

223-233

234-244

245-254

255-270

Enhancement of alpha amylase production by Aspergillus flavus AUMC 11685 on mandarin (Citrus reticulata) peel using submerged fermentation

Ahmed Mohammed Rawaa, Esam H. Ali, Mohamed A. El-Nagdy, Saleh M. Al-Garni, Saleh M. Ahmed, Ahmed M. Rawaa

Influence of extracellular matrix on the proliferation and adhesion properties of stem cells derived from different sources

Anna Bajek, Dorota Porowińska, Krzysztof Roszkowski

Functional assessments and histopathology of hepatorenal tissues of rats treated with raw and processed herbs

Okey A. Ojiako, Paul Chidoka Chikezie, Doris I. Ukairo, Chiedozie O. Ibegbulem, Reginald N. Nwaoguikpe

In vitro regeneration of plantlets from nodal explants of Aristolochia saccata and Aristolochia cathcartii

Bhaskar Sarma, Bhaben Tanti

Proposal for screening of kidney disease in a random population based on World Kidney Day campaign

Marcelo Bacci, Victor Jordão, Livia Vasconcelos, Thiago Castanheira, Ronaldo Bergamo, Daniel Santos, Ana Carolina Mottecy, Ligia Azzalis, Edimar Pereira, Beatriz Alves, Fernando Fonseca

Incidence and significance of black aspergilli in agricultural commodities: a review, with a key to all species accepted to-date

Mady A. Ismail

Biological action of Piper nigrum - the king of spices

Arun Kumar Srivastava

In vitro assessment of antimicrobial and anti-inflammatory potential of endophytic fungal metabolites extracts

A. M. Moharram, A. A. Zohri, Hossam El-Din M. Omar, O. A. Abd El-Ghani

Chrysin and its potential antineoplastic effect

Patrycja Chylińska-Wrzos, Marta Lis-Sochocka, Barbara Jodłowska-Jędrych

Managing phosphorus in terrestrial ecosystem: a review

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of Biological Research

European Journal of Biological Research 2017; 7 (3): 154-164

Enhancement of alpha amylase production by

Aspergillus

flavus

AUMC 11685 on mandarin (

Citrus reticulata

) peel

using submerged fermentation

Esam H. Ali¹, Mohamed A. El-Nagdy¹, Saleh M. Al-Garni², Mohamed S. Ahmed²,

Ahmed M. Rawaa¹*

¹ Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut, Egypt

² Microbiology Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia *Corresponding author: Ahmed M. Rawaa; E-mail: ahmedrawaa@hotmail.com

ABSTRACT

Mandarin peel as submerged fermentation (SmF) source was tested for the production of alpha amylase enzyme by strain of Aspergillus flavus AUMC 11685. Incubation period, concentration of substrate, temperature, pH and size of inoculum were optimized to achieve the maximum production of alpha amylase enzyme by Aspergillus flavus using mandarin peel. The maximum production of alpha amylase enzyme by Aspergillus flavus was recorded at 4-5 days of incubation, 3% substrate concentration, inoculum concentration 10%, temperature 28-40°C and pH 4-5.5.

Keywords: Mandarin; α-amylase; Aspergillus flavus; Submerged fermentation.

1. INTRODUCTION

Nowadays, the new potential of using micro-organism as biotechnological source of industrially relevant enzymes has stimulated interest in exploration of extracellular enzymatic activities in several microorganisms [1-3]. Enzymes have been

used for thousands of years to produce food and beverages, such as cheese, yoghurt, beer and wine [4].

Enzymes are protein catalysts synthesized by living systems and are important in synthetic as well as degradative process. Alpha amylase enzyme (α-1,4 glucan-glucanohydrolase) is widely distri-buted in nature. This extracellular starch degrading enzyme hydrolyses α-1,4 glucosidic linkages ran-domly throughout the starch molecule in an endo-fashion producing oligosaccharides and mono-saccharides including maltose, glucose and alpha limit dextrin [5-8]. Alpha-amylase enzymes account 65% of enzyme market in world. Amylases had numerous applications including liquefaction of starch in the traditional beverages, baking and textile industry for desizing of fabrics [9-11]. Moreover, they have been applied in paper manufacture, medical fields as digestive and as detergent additives [12, 13] . Hence, any substantial reduction in the cost of production of enzymes will be a commercial positive stimulus [4]. Fungi are particularly interesting due to their easy cultivation, and high production of extracellular enzymes of large industrial potential. These enzymes have Received: 14 March 2017; Revised submission: 08 June 2017; Accepted: 22 June 2017

Copyright: © The Author(s) 2017. European Journal of Biological Research © T.M.Karpiński 2017. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits

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European Journal of Biological Research 2017; 7 (3): 154-164 commercial application in various industries [14].

Many useful enzymes are produced using industrial fermentation belonging to the genus Aspergillus [15, 16]. In fact Aspergillus niger is the largest fungal source of enzymes [17, 18].

α-amylase is widespread in animals, fungi, plants, and are also found in bacteria [19, 20]. Amylases from microbial sources are generally used in industrial processes due to a number of factors including productivity, thermostability of the enzy-me as well as ease of cultivating microorganisms [21]. Alpha-amylases are produced commercially in bulk from microorganisms and represent about 25-33% of the world enzyme market [22].

Many attempts have been made to optimize culture conditions and suitable strains of fungi [23]. Selection of the microbial source for α-amylase production depends on several features, such as the type of culture (solid-state or submerged fermen-tation), pH and genotypic characteristic of the strain [24].

Fermentation is the technique of biological conversion of complex substrates into simple compounds by various microorganisms such as bacteria and fungi. Several additional compounds also released apart from the usual products of fermentation called secondary metabolites which, range from several antibiotics to enzymes [25, 26]. The development of techniques such as Solid State Fermentation (SSF) and Submerged Fermentation (SmF) has lead to industrial-level production of useful enzymes. Submerged fermentation utilizes free flowing liquid substrates, such as broths, enzymes are secreted into the fermentation broth [27]. The purification of products is easier in SmF. More than 75% of the industrial enzymes are produced using SmF, one of the major reasons being that SmF supports the utilization of genetically modified organisms to a greater extent than SSF. Another reason why SmF is widely used is the lack of paraphernalia regarding the production of various enzymes using SSF. This is highly critical due to the fact that the metabolism exhibited by microorga-nisms is different in SSF and SmF [28]. Solid-state fermentation (SSF) has been defined as the fermen-tation process which involves solid matrix and is carried out in absence or near absence of free water. The solid matrix could be either the source of nutrients or simply a support supplemented by the

suitable nutrients that allows the development of the microorganisms [29]. There are some disadvantages of SSF like difficulties on scale-up, low mix effectively, difficult control of process parameters (pH, heat, moisture, nutrient conditions), problems with heat build-up, higher impurity product and increasing recovery product costs [30]. Optimization of various parameters is one of the most important techniques used for the production of enzymes in large quantities to meet industrial demands [31]. Production of extracellular alpha-amylase in fungi is known to depend on the growth of mycelium and both morphological and metabolic state of the culture [32].

The selection of a substrate (agricultural waste) for enzyme production depends upon several factors mainly related with cost and availability of the substrate, the solid substrate not only supplies the nutrients to the microbial culture growing in it but also serves as anchorage for the cells [33]. These agriculture wastes consist of carbon and nitrogen sources necessary for the growth and metabolism of microorganisms [34, 35]. These nutrient sources included orange and mandarin wastes, rice and wheat bran, tea waste, cassava flour, oil palm waste, apple pomace and banana waste [36].

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European Journal of Biological Research 2017; 7 (3): 154-164 researches were done studying the production of

enzymes by fungal strains using mandarin peel wastes. This work aims to evaluate the potentials of Aspergillus flavus strain AUMC 11685 isolated from accumulated rains water at Jeddah region to produce extracellular alpha amylase enzyme using mandarin peel wastes as substrate by submerged fermentation. Moreover, several factors including: pH, temperature, incubation period and concen-tration of each of raw material and inoculum were tested for optimization and enhancement of

α-amylase enzyme production by Aspergillus flavus AUMC 11685 using mandarin peel wastes as a substrate in the submerged fermentation process.

2. MATERIALS AND METHODS

2.1. Microorganism

Pure culture of Aspergillus flavus AUMC 11685, which was isolated from accumulated rains water, Jeddah, Saudi Arabia, was grown and maintained on potato dextrose agar and it used as an inoculum during optimization steps of the study. The identification of the tested fungal species was confirmed by Assiut University Mycological Centre (AUMC) and the strain is deposited at Assiut University Mycological Centre under the code Aspergillus flavus AUMC 11685. The slants of the strain were grown at 28°C for seven days and stored at 4°C.

2.2. Agriculture wastes

Five grams of the agricultural waste; mandarin peel were mixed in 500 ml Erlenmeyer conical flasks containing 100 ml distilled water and sterilized in autoclave at 121°C for 20 min. Mandarin peel chosen as the sole nutrient source for submerged fermentation (SmF).

2.3. Optimization methodology of submerged fermentation (SmF)

Submerged fermentation was performed to study the effect of various physico-chemical parameters required for the optimum production of

α-amylase enzyme by A. flavus AUMC 11685. Conidia are scrapped from mycelia of the terrestrial

fungal species which are grown on slants for five days at 28°C and suspended in sterile distilled water. One ml of this suspension is used to inoculate, under aseptic conditions, Erlenmeyer flasks (500 ml capacity) each containing 100 ml of previous sterilized medium (agriculture waste medium). The inoculated flasks are incubated at 28°C on a rotary shaker at 160 rpm for 7 days (Figure 1). Aspergillus flavus was subjected to several optimization factors for enhancement of

α-amylase enzyme production using mandarin peel wastes by SmF. Each experiment was done in thrice.

Figure 1. The inoculated flask containing the submerged

fermentation medium of mandarin peel wastes.

2.3.1. Initial pH

The tested fungal strain of Aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different initial pH 2, 4, 5.5, 7 and 10. The initial pH was adjusted by 0.1 M HCl or 0.1 M NaOH. The assay of α-amylase produced was determined.

2.3.2. Incubation temperature

The tested fungal strain of Aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different incubation temperature degrees 20, 25, 28, 35, 40 and 50°C at the optimum initial pH. The assay of α-amylase produced was determined.

2.3.3. Incubation period

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European Journal of Biological Research 2017; 7 (3): 154-164 was grown on mandarin peel medium by applying

the previously mentioned fermentation process at several intervals of inoculation periods 2, 3, 4, 5, 6 and 7 days at both the optimum temperature and initial pH. The assay of α-amylase produced was determined.

2.3.4. Concentration of raw material

The tested fungal strain of Aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different concentration of raw material of mandarin peel waste 1, 3, 5, 7 and 9 g at the optimum temperature, initial pH and the optimal incubation period. The assay of α-amylase produced was determined.

2.3.5. Concentration of inoculum

The tested fungal strain of Aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different inoculum concentrations 0.5, 1, 2, 5 and 10 ml at the optimum temperature, initial pH, the optimal incubation period and raw material concentration. The assay of α-amylase produced was determined.

2.4. Partially purification of enzymes

Conical flasks containing the agriculture waste medium and the fungal inocula are filtered at the end of the incubation period. Then, the filtrate introduced into dialysis bag against distilled water for 24 hours. The dialyzed filtrate was centrifuged at 10,000 rpm for 20 min. The supernatant was pooled and designated as cell-free broth. The cell free broth was frozen at -20°C for further purification steps [41].

2.5. Enzyme assay

α-amylase activity was determined by measurement of glucose released from starch according to the method of Miller [42]. The reaction mixture in tubes contained 125 µl soluble potato starch 0.2%, 125 µl sodium acetate buffer, pH 5.5, 50 µl of enzyme solution and distilled

water to give a final volume of 0.5 ml (test solution) and was incubated at 37°C for 30 min. The reaction was stopped by the addition of 0.5 ml dinitrosalicylic acid reagent (DNS), followed by incubation in a boiling water bath for 10 min followed by cooling. The absorbance was recorded at 560 nm. The enzymatically liberated reducing sugar was calculated from a standard curve using glucose. One unit of enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions.

3. RESULTS

Alpha-amylase production by Aspergillus flavus AUMC 11685 isolated from water habitats in Jeddah, Saudi Arabia using mandarin peel by submerged fermentation was optimized.

3.1. The effect of pH

The result of the effect of different pH values on the production of α-amylase by Aspergillus flavus AUMC 11685 was shown in Table 1. The lowest productivity was obtained at pH 2 (7.32 U/ml), then the α-amylase activity sharply increased at pH 4 (24.73 U/ml), and gradually increased at pH 5.5 (26.90 U/ml). At pH values higher than 5.5 the productivity sharply decreased at pH 7 (17.99 U/ml) and at alkaline pH 10 (17.03 U/ml). The highest α-amylase enzyme production was recorded at pH 5.5.

3.2. The effect of incubation temperature

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European Journal of Biological Research 2017; 7 (3): 154-164 Table 1. Effect of different pH values on α-amylase

production (U/ml) by Aspergillus flavus isolated from water habitats in Saudi Arabia using mandarin peel wastes as submerged culture.

pH values Extracellular α-amylase production (U/ml)

2 7.32

4 24.73

5.5 26.90

7 17.99

10 17.03

One unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions.

Table 2. Effect of different incubation temperatures on α -amylase production (U/ml) by Aspergillus flavus isolated from water habitats in Saudi Arabia using mandarin peel wastes as submerged culture.

Incubation temperatures

Extracellular α-amylase production (U/ml)

20 ºC 15.76 25 ºC 18.24 28 ºC 26.90 35 ºC 18.36 40 ºC 18.67 50 ºC 14.38

One unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions.

3.3. The effect of different concentrations of substrate (mandarin peel)

The result of the effect of different concentrations of mandarin peel medium on the production of α-amylase was shown in Table 3. Our results showed that A. flavus could produce small amount of α-amylase using mandarin peel medium at concentration 1% (g/100 ml) (12.82 U/ml), then pointedly increased to the highest yield at concen-tration 3% (28.28 U/ml) and slightly decreased at concentration 5% (26.90 U/ml). After this, the productivity decreased gradually at concentrations 7% (17.24 U/ml) and 9% (16.79 U/ml).

Table 3. Effect of different concentrations of mandarin

peel medium on α-amylase production (U/ml) by Aspergillus flavus isolated from water habitats in Saudi Arabia using mandarin peel wastes as submerged culture.

Concentration of mandarin peel medium

Extracellular α-amylase

production (U/ml)

1 g 12.82 3 g 28.28 5 g 26.90 7 g 17.24 9 g 16.79

One unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions.

3.4. The effect of incubation period

Alpha-amylase production was detected at different incubation periods as shown in Table 4. Aspergillus flavus could start α-amylase production using mandarin peel medium after two days of incubation (13.46 U/ml) and then the productivity increased in gradual trend at three days of incubation (18.10 U/ml). α-amylase production sharply increased recording the peak rate at the fourth day of incubation (33.52 U/ml), then progressively decreased in gradual trend at five (28.93 U/ml), six (27.12 U/ml) and seven (26.90 U/ml) days of incubation. The highest α-amylase enzyme production was obtained after incubation for 4 days.

3.5. The effect of inoculum concentration

The result of the effect of different concen-trations of A. flavus inoculum on the production of

α-amylase was displayed in Table 5. Little output of

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European Journal of Biological Research 2017; 7 (3): 154-164 Table 4. Effect of different Incubation periods on α

-amylase production (U/ml) by Aspergillus flavus isolated from water habitats in Saudi Arabia using mandarin peel wastes as submerged culture.

Incubation periods Extracellular α-amylase production (U/ml)

2 days 13.46 3 days 18.10 4 days 33.52 5 days 28.93 6 days 27.12 7 days 26.90

One unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions.

Table 5. Effect of different Inoculum concentrations on

α-amylase production (U/ml) by Aspergillus flavus isolated from water habitats in Saudi Arabia using mandarin peel wastes as submerged culture.

Inoculum concentration

Extracellular α-amylase

production (U/ml)

0.5 ml 3.04 1 ml 26.90 2 ml 30.04 5 ml 35.73 10 ml 64.30

One unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions.

4. DISCUSSION

The production of α-amylase using submer-ged fermentation by fungi has been reported by many workers [43-46]. In the present study, the optimum conditions for α-amylase production by Aspergillus flavus were acidic pH range 4-5.5, a temperature of 25-40°C for a period of 4-5 days using concentration of mandarin peels medium 3-5% and the concentration of A. flavus microbial suspension was positively related with productivity.

From our results extracellular α-amylase could be produced by A. flavus using mandarin peels at all pH values used but with different amounts. Extreme pH values (highly alkaline or

acidic) decreased α-amylase production. At tempe-rature 28°C, A. flavus showed the maximum

α-amylase production, whereas below or above this temperature α-amylase production declined gradu-ally. Extracellular α-amylase could be produced by A. flavus using mandarin peels (concentration 1%) and increased at concentration 3%, above this concentration there was a negative relation between

α-amylase productivity and concentration of manda-rin peels medium. After 4 incubation days A. flavus showed the maximum α-amylase production, whereas at less than this the α-amylase production declined or more than 4 days the productivity declined gradually. There was positive relation between concentration of A. flavus microbial sus-pension and α-amylase production. Our study reported that the highest α-amylase enzyme production by A. flavus isolated from water habitats in Saudi Arabia using mandarin peels medium was recorded at pH 5.5, temperature 28°C and incu-bation period of 4 days. The maximum productivity of α-amylase was detected when using concen-tration 3 g/100 ml of mandarin peels medium and 10% concentration of A. flavus microbial suspen-sion.

Among the physical parameters, the pH of medium plays an important role by inducing morphological changes in fungi and in enzyme secretion [47]. The synthesis of extracellular

α-amylase is affected by the pH [48].

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European Journal of Biological Research 2017; 7 (3): 154-164 al. [54] reported that pH=4.0 to be the best for

the production of α-amylase by A. awamori. With inconsistence of our results Suganyadevi et al. [55] reported that the maximum production of α-amylase by A. niger on tuber of Ipomoea batatas was attai-ned at pH 7. Moreover, Varalakshmi et al. [56] and Arunsasi et al. [8] found that the highest production of α-amylase by Aspergillus flavus on wheat bran and Cocos nucifera meal was accomplished at pH 7.5.

Temperature is one of the important factors, which strongly affect alpha amylase production by fermentation process [19, 57, 58]. Our findings were compatible with Suganyadevi et al. [55] who observed that the maximum yield of α-amylase production by A. niger was possible by submerged fermentation supplied with tuber of Ipomoea batatas at room temperature (28°C). Our results are also similar to those obtained by Ramachandran et al. [59] who studied α-amylase enzyme synthesis by Aspergillus oryzae on coconut oil cake and reported that 30°C proved to be the best temperature for the enzyme synthesis. In addition, similar results were obtained by Arunsasi et al. [8] who studied

α-amylase enzyme production by Aspergillus flavus on Cocos nucifera meal.

Incubation at higher temperature affected the fungus harmfully. In agreement of our output Sivaramakrishnan et al. [49] reported that alpha amylase enzyme synthesis by Aspergillus oryzae occurred between 20-45°C with an optimum at 30°C on wheat bran. Acourene et al. [47] reported that alpha-amylase production by Candida guilliermondii on date wastes was low at 20°C, and increased to a maximum at 30°C. A further increment in tempe-rature resulted in a decrease in dry biomass and

α-amylase production. At higher temperature, due to the production of large amount of metabolic heat, the fermenting substrate temperature shoots up, thereby inhibiting microbial growth and enzyme formation [60]. Temperature above 45°C results in moisture loss of the substrate, which affects metabolic activities of fungi, and results in reduced growth and α-amylase production [61]. However, Kunameni et al. [62] and Ravi et al. [63] reported that optimum temperature for amylase production by Trichoderma lanuginosus and Humicola lanu-ginosa is 50°C. Moreover, the optimum temperature for the maximum α-amylase activity by some

Aspergillus spp. was 30°C [34, 45, 46, 50, 51] and also the same by Penicillium brevicompactum [64] and Penicillium purpurogenum [52].

Regarding the impact of incubation period on alpha amylase production, our findings were nearly came in agreement with Kareem et al. [36] who reported that the maximum α-amylase production by Aspergillus oryzae on Cowpea wastes was recorded after 72 hours of incubation. Sivaramakrishnan et al. [49] also reported the same during on wheat bran and Acourene et al. [47] with Candida guillier-mondii on date wastes. In contrast to our results, Silva et al. [52] observed the highest production by Penicillium purpurogenum and Penicillium brevi-compactum after 6 and 7 days of incubation and Balkan and Ertan [64] after 7 days with Penicillium brevicompactum.

No doubt that concentration of substrate affects α-amylase production. Similar to our find-ings Mohamed et al. [41] who studied the effect of mandarin peel concentration on α-amylase produc-tion by Trichoderma harzianum found that the highest level of enzyme activity was obtained at 5% of mandarin peel. Further concentration of mandarin peel repressed the enzyme production. Ramachan-dran et al. [59] reported that 0.5% concentration of starch was most suitable and higher concentrations of starch resulted in the inhibition of α-amylase enzyme synthesis by Aspergillus oryzae (data not shown).

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European Journal of Biological Research 2017; 7 (3): 154-164 gave the maximum production of alpha-amylase.

Kareem et al. [36] reported that the maximum amylase production of α-amylase enzyme is attained at 4% Aspergillus oryzae inoculum level on Cowpea wastes and a further increase in the inoculums size did not increase the amylase yield. A lower level of inoculum may not be sufficient for initiating growth and enzyme synthesis.

General outlook indicates that our results are promising in enhancement of alpha-amylase production by growing strain of Aspergillus flavus AUMC 11685 on mandarin peel wastes in submerged culture fermentation. Based on the results obtained, mandarin peel wastes and our strain of Apergillus flavus were nearly more efficient in the quantity of alpha amylase production at the optimal conditions when they were compared with other wastes or substrates and microorganism in reported previous works. We have obtained 64.30 U/ml whereas Balkan and Ertan [64] detected 40 U/ml on rye straw, 50 U/ml on wheat straw, 25 U/ml on wheat branand 160 U/ml on corncob leaf by Penicillium chrysogenum, Farid and Shata [65] detected 1362.09 IU/g on wheat flour by Aspergillus oryzae LS1, Acourene et al. [47] estimated 1519.23 μmol/l/min on date wastes by Candida guilliermondii CGL-A10, Hang and Woodams [66] harvested 29 U/ml on baked-bean wastes and 0.06 U/ml on 2% cornmeal by Aspergillus foetidus NRRL 337, Suganthi et al. [67] found 43 U/mg on groundnut oil cake by Aspergillus niger BAN 3E, Singh et al. [27] indicated 11.0 U/ml on bacteriological peptone, MgSO4·7H2O, KCl, starch by Bacillus sp., Krishna et al. [68] evaluated 23 U/ml on banana peel by Aspergillus niger NCIM 616 and Kumar et al. [69] produced 90 U/ml on sweet lime peel by Aspergillus niger.

5. CONCLUSION

The present study reveals that mandarin peel waste can be used safely as optional substrates than other agricultural/agro-industrial wastes such as wheat, corn, rice, potato and apple for the production of α-amylase enzyme. This study established the potential of the fungal strain of Aspergillus flavus AUMC 11685 for economic α-amylase production on mandarin peel in optimum conditions. This work gives an insight into the exploitation of a new agriculture wastes for the

production of some industrial enzymes in appreciable levels.

AUTHORS’ CONTRIBUTION

All the authors contributed in the success of this research article. The final manuscript has been prepared and revised by EHA and AMR. The final manuscript has been read and approved by all authors.

TRANSPARENCY DECLARATION

The authors declare no conflicts of interest.

REFERENCES

1. Bilinski CA, Stewart GC. Production and characterization of α-amylase from Aspergillus niger. Int J Eng Sci Tech. 1995; 18: 551-556. 2. Akpan I, Bankjole MO, Adesermowo AM.

Production of α-amylase by Aspergillus niger in a cheap solid medium using rice bran and agricultural material. Braz Arch. Biol Technol 1999; 44: 79-88. 3. Buzzini P, Martini A. Extracellular enzymatic

activity profiles in yeast and yeast like strains isolated from tropical environments. J Appl Microbiol. 2002; 93: 1020-1025.

4. Renge VC, Khedkar SV, Nandurkar R. Enzyme synthesis by fermentation method. SRCC. 2012; 2(4): 585-590.

5. Omemu AM, Akpan I, Bankole MO, Teniola OD. Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil. Afr J Biotechnol. 2005; 4(1): 19-25.

6. Bhanja T, Rout S, Banerje, R, Bhattacharya BC. Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor. Bioprocess Biosyst Eng. 2007; 30: 369-376.

7. Leman P, Goesaert H, Delcour JA. Residual amylopectin structures of amylase treated wheat slurries reflect amylase mode of action. Food Hydrocolloids. 2009; 23(1): 153-164.

8. Arunsasi, ManthiriKani S, Jegadeesh G, Ravikumar M. Submerged fermentation of amylase enzyme by Aspergillus flavus using Cocos nucifera meal. Kathmandu Univ J Sci Eng Tech. 2010; 6: 75-87. 9. Dauter Z, Dauter M, Brzozowski AM, Christensen S,

(12)

European Journal of Biological Research 2017; 7 (3): 154-164

10. Hendriksen H, Pedersen S, Bisgard-Frantzen H. A process for textile warp sizing using enzymatically modified starches. Patent Application. 1999; WO: 99/35325.

11. Nielsen JE, Borchert TV. Protein engineering of bacterial α-amylases review. Biochim Biophys Acta. 2000; 1543: 253-274.

12. Bruinenberg P, Hulst A, Faber A, Voogd R. A process for surface sizing or coating of paper. Eur Patent Application. 1996; 690,170 A1.

13. Mitidieri S, Martinelli AHS, Schrank A, Vainstein MH. Enzymatic detergent formulation containing amylase from Aspergillus niger: a comparative study with commercial detergent formulations. Biores Technol. 2006; 97: 1217-1224.

14. Mishra BK, Dadhich SK. Production of amylase and xylanase enzymes from soil fungi of Rajasthan. JASR. 2010; 1(1): 21-23.

15. Ugru GC, Akinayanju JA, Sani A. The use of yam peel for growth of locally isolated Aspergillus niger and amylase production. Enzyme Microb Technol. 1997; 21: 48-51.

16. Holker U, Hofer M, Lenz J. Biotechnological advantages of laboratory-scale solid state fermentation with fungi. Appl Microbiol Biotechnol. 2004; 64: 175-186.

17. Perrone G, Mulè G, Susca A, Battilani P, Pietri A, Logrieco A. Ochratoxin A production and AFLP analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger strains isolated from grapes in Italy. Appl Environ Microbiol. 2006; 72: 680-685.

18. Tjamos SE, Antoniou PP, Kazantzidou A, Antonopoulos DF, Papageorgiou I, Tjamos EC. Aspergillus niger and Aspegillus carbonarius in Corinth raisin and wine-producing vineyards in Greece: population composition, ochratoxin A production and chemical control. J Phytopathol. 2004; 152: 250-255.

19. Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation. Process Biochem. 2000; 35: 1153-1169.

20. Da Lagea JL, Etienn GJ, Danchinc EGJ, Casane D. Where do animal α-amylases come from? FEBS J. 2007; 581: 3927-3935.

21. Reddy R, Reddy G, Seenayya G. Enhanced production of thermostable α-amylase of pullulunase in the presence of surfactants by Clostridium thermosulfurogenes SV2. Process Biochem. 1999; 34: 87-92.

22. Nguyen QD, Rezessy-Szabo JM, Claeyssens M, Stals I, Hoschke A. Purification and characterization of amylolytic enzymes from thermophilic fungus

Thermomyces lanuginosus strain ATCC 34626. Enzyme Microb Technol. 2002; 31: 345-352.

23. Abu EA, Ado SA, James DB. Raw starch degrading amylase production of mixed culture of Aspergillus niger and Saccharomyces cerevisiae grown on Sorghum pomace. Afr J Biotechnol. 2005; 4: 785-790.

24. Khairnar Y, Krishna K, Boraste A, Gupta N, Trivedi S, Patil P, et al. Study of pectinase production in submerged fermentation using different strains of Aspergillus niger. Int J Microbiol Res. 2009; 1(2): 13-17.

25. Machado CM, Oishi BO, Pandey A, Soccol CR. Kinetics of Gibberella fujikori growth and gibberellic acid production by solid state fermentation in a packed-bed column bioreactor. Biotechnol Prog. 2004; 20: 1449-1453.

26. Robinson T, Singh D, Nigam P. Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl Microbiol Biotechnol. 2001; 55: 284-289.

27. Singh P, Gupta P, Singh R, Sharma R. Factors affecting alpha amylase production on submerged fermentation by Bacillus sp. IJPLS. 2012; 3(12): 2243-2246.

28. Subramaniyam R, Vimala R. Solid state and submerged fermentation for the production of bioactive substances: a comparative study. Int J Sec Nature. 2012; 3(3): 480-486.

29. Singhania R, Patel A, Soccolc C, Pandeya A. Recent advances in solid-state fermentation. Biochem Eng J. 2009; 44: 13-18.

30. Couto S, Sanroman M. Application of solid-state fermentation to food industry - a review. J Food Eng. 2005; 76: 291-302.

31. Tanyildizi MS, Ozer D, Elibol M. Optimization of alpha-amylase production by Bacillus sp. using response surface methodology. Process Biochem. 2005; 40: 2291-2296.

32. Carlsen M, Spohr A, Nielsen J, Villadsen J. Morphology and physiology of an α-amylase producing strain of Aspergillus oryzae during batch cultivations. Biotechnol Bioeng.1996; 49: 266-276. 33. Nimkar MD, Deogade NG, Kawale M. Production of

alpha-amylase from Bacillus subtilis & Aspergillus niger using different agro waste by solid state fermentation. Asia J Biotech Res. 2010; 01: 23-28. 34. Djekrif-Dakhmouche S, Gheribi-Aoulmi Z, Meraihi

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European Journal of Biological Research 2017; 7 (3): 154-164

35. Haq I, Ashraf H, Qadeer MA, Iqbal J. Pearl millet, a source of alpha amylase production by Bacillus licheniformis. Biores Technol. 2005; 96: 1201-1204. 36. Kareem SO, Akpan I, Oduntan SB. Cowpea waste: a

novel substrate for solid state production of amylase by Aspergillus oryzae. Afr J Microbiol Res. 2009; 3(12): 974-977.

37. Tomsen MH. Complex media from processing of agricultural crops for microbial fermentation. Appl Microbiol Biotech. 2005; 68: 598-606.

38. Garzón CG, Hours RA. Citrus waste: an alternative substrate for pectinase production in solid-state culture. Biores Technol. 1992; 39: 93-95.

39. Rafiq S, Kaula R, Sofia SA, Bashira N, Nazirb F, Nayikc G. Citrus peel as a source of functional ingredient: a review. J Saudi Soc Agric Sci. 2016; In press.

40. Mamma D, Kourtoglou E, Christakopoulos P. Fungal multienzyme production on industrial by-products of the citrus processing industry. Biores Technol. 2008; 99: 2373-2383.

41. Mohamed S, Azhar E, Ba-Akdah M, Tashkandy N, Kumosani T. Production, purification and characterization of α-amylase from Trichoderma harzianum grown on mandarin peel. Afr J Microbiol Res. 2011; 5(8): 930-940.

42. Miller GL. Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal Chem. 1959; 31: 426-429.

43. Hernandez MS, Rodrıguez MR, Perez-Guerra N, Perez-Roses R. Amylase production by Aspergillus niger in submerged cultivation on two wastes from food industries. J Food Eng. 2006; 73: 93-100. 44. Kathiresan K, Manivannan S. Amylase production by

Penicillium fellutanum isolated from mangrove rhizosphere soil. Afr J Biotechnol. 2006; 5(10): 829-832.

45. Lagzouli M, Charouf R, El-Yachioui O, Berny MEH, Jadal M. Optimization de la croissance et de la production de gluco amylase extra cellulaire par Candida guilliermondii. Bull Soc Pharmacie. 2007; 70: 146-251.

46. Wang Q, Wang X, Maa H. Glucoamylase production from food wastes by Aspergillus niger under submerged fermentation. Process Biochem. 2008; 43: 280-286.

47. Acourene S, Amourache L, Benchabane A, Djaafri K. Utilisation of date wastes as substrate for the production of α-amylase. Int Food Res J. 2013; 20(3): 1367-1372.

48. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial α-amylases: a biotechnolo-gical perspective. Process Biochem. 2003; 38: 1599-1616.

49. Sivaramakrishnan S, Gangadharan D, Nampoothiri K, Soccol C, Pandey A. Alpha amylase production by Aspergillus oryzae employing soild-state fermentation. J Sci Ind Res. 2007; 66: 621-626. 50. Alva S, Anupama J, Savla J, Chiu, YY, Vyshali P,

Shruti M, et al. Production and characterization of fungal amylase enzyme isolated from Aspergillus sp. JGI 12 in solid state culture. Afr J Biotechnol. 2007; 6(5): 576-581.

51. Renato PR, Nelson PG. Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material. World J Microbiol Biotechnol. 2009; 25(11): 1929-1939.

52. Silva T, Oliveira M, Somera A, Jorge J, Terenzi H, Lourdes M, et al. Thermostable saccharogenic amylase produced under submerged fermentation by filamentous fungus Penicillium purpurogenum. Braz J Microbiol. 2011; 42: 1136-1140.

53. Guillen-Moreira F, Arrias de Lima F, Fazzano-Pedrinho SR, Lenartovicz V, Giatti-Marques de Souza F, Peralta RM. Production of amylases by Aspergillus tamarii. Rev Microbiol. 1999; 30(2): 1-9. 54. Pavezzi FC, Gomes E, Roberto-Da-Silva R. Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources. Braz J Microbiol. 2008; 39(1): 127-135. 55. Sundar R, Liji T, Rajila C, Suganyadevi P. Amylase

production by Aspergillus niger under submerged fermentation using Ipomoea batatas. Int J Appl Biol Pharmac Technol. 2012; 3(1): 175-182.

56. Varalakshmi KN, Kumudini BS, Nandini BN, Solomon J, Suhas R, Mahesh B, Kavitha AP. Production and characterization of alpha amylase from Aspergillus niger JGI 24 isolated in Bangalore. Pol J Microbiol. 2009; 58(1): 29-36.

57. Pandey A. Production of starch saccharifying enzyme (glucoamylase) in solid cultures. Starch. 1992; 44: 75-77.

58. Vidyalakshmi R, Paranthaman R, Indhumathi J. Amylase production on submerged fermentation by Bacillus spp. World J Chem. 2009; 4(1): 89-91. 59. Ramachandran S, Patel A, Nampoothiri K, Francis

F, Nagy V, Szakacs G, Pandey A. Coconut oil cake - a potential raw material for the production of α -amylase. Biores Technol. 2004; 93: 169-174.

60. Nawaz-Bhatti H, Hamid-Rashid M, Nawaz R, Asgher M, Perveen M, Abdul-Jabbar A. Optimization of media for enhanced glucoamylase production in solid-state fermentation by Fusarium solani. Food Technol Biotechnol. 2007; 45(1): 51-56. 61. Sindhu R, Suprabha GN, Shashidhar S. Optimization

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-European Journal of Biological Research 2017; 7 (3): 154-164

amylase from Penicillium janthinellum (NCIM 4960) under solid state fermentation. Afr J Microbiol Res. 2009; 3(9): 498-503.

62. Kunameni A, Permaul K, Singh S. Amylase production in solid state fermentation by the thermophilic fungus Thermomyces lanuginosus. J Biosci Bioeng. 2005; 100: 168-171.

63. Ravi KS, Shashi K, Surendra K. Production of α -amylase from agricultural by products by Humicola lanuginosa in solid state fermentation. Curr Trends Biotechnol Pharm. 2009; 3(2): 172-180.

64. Balkan B, Ertan F. The production of a new fungal alpha-amylase degraded the raw starch by means of solid-state fermentation. Prep Biochem Biotechnol. 2010; 40(3): 213-228.

65. Farid MA, Shata HM. Amylase production from Aspergillus oryzae LS1 by solid-state fermentation and its use for the hydrolysis of wheat flour. Iran J Biotech. 2011; 9(4): 267-274.

66. Hang Y D, Woodams EE. Baked-bean waste: a potential substrate for producing fungal amylases. Appl Environ Microbiol. 1977; 33(6): 1293-1294. 67. Suganthi R, Benazir JF, Santhi R, Ramesh

K, Anjana H, Nitya M, et al. Amylase production by Aspergillus niger under solid state fermentation using agroindustrial wastes. IJEST. 2011; 3(2): 1756-1763.

68. Krishna PR, Sirvastava AK, Ramaswamy NK, Suprasanna P, Sonaza SFD. Banana peel as a substrate for amylase production using Aspergillus niger NCIM 616. IJBT. 2012; 11: 314-319.

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of Biological Research

European Journal of Biological Research 2017; 7 (3): 165-171

Influence of extracellular matrix on the proliferation

and adhesion properties of stem cells derived from

different sources

Anna Bajek

1

, Dorota Porowińska

2

, Krzysztof Roszkowski

3

*

¹ Department of Tissue Engineering, Nicolaus Copernicus University, Collegium Medicum Bydgoszcz, Poland

2

Department of Biochemistry, Nicolaus Copernicus University, Toruń, Poland

3

Department of Oncology, Radiotherapy and Oncological Ginecology, Nicolaus Copernicus University, Romanowskiej 2, 85-796 Bydgoszcz, Poland

*Corresponding author: Prof. Krzysztof Roszkowski; Tel. +48 523743744; E-mail: roszkowskik@cm.umk.pl

ABSTRACT

One of the most important issues in regenerative medicine is the development of culture conditions mimicking the natural ones, which allows obtaining a large number of cells and their long-term maintenance in undifferentiated state. In vivo, cells are surrounded by a specific microenvironment called extracellular matrix (ECM), which plays an important role in the regulation of processes such as proliferation, migration, differentiation or apoptosis. In this study we assessed the influence of different extracellular matrix components (fibronectin, laminin, collagen IV, poly-D-lysine) on the in vitro adhesion and proliferation of stem cells isolated from bone marrow, adipose tissue and hair follicles. Our results showed that stem cells derived from different sources present various responses to ECM components. None of the tested extracellular proteins reduced the proliferation of bone marrow as well as adipose-derived mesenchymal stem cells, with the exception of laminin. This demonstrates the biocompatibility of such modified surfaces and possibility of using them for culturing these types of stem cells. Different results were obtained for hair

follicle stem cells. The presented results indicate that ECM is an important component of the cellular niche in the tissue. It is also possible that ECM is required for the reconstitution of the niche of stem cells in vitro.

Keywords: Stem cells; Bone marrow; Adipose tissue; Hair follicles; Extracellular matrix.

1. INTRODUCTION

Tissue engineering methods offer new possibilities for the regeneration of diseased and damaged tissues and thus find an increasing attention in clinical practice. In tissue engineering, a variety of different cell types are used. However, the most attractive type are stem cells, particularly mesenchymal stem cells (MSCs).

One of the most important issues in the use of stem cells is the development of culture conditions mimicking the natural ones, which allows obtaining a large number of cells and their long-term maintenance in undifferentiated state. The proper growth and functioning of cells in vivo and in vitro depends on many factors, which result not only Received: 09 March 2017; Revised submission: 26 June 2017; Accepted: 29 June 2017

Copyright: © The Author(s) 2017. European Journal of Biological Research © T.M.Karpiński 2017. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits

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from the interaction between cells (cell-cell type), but also from the interaction between cells and the extracellular environment (cell-matrix type) [1-3]. In vivo, cells are surrounded by a specific micro-environment called extracellular matrix (ECM),

which plays an important role in the regulation of processes such as proliferation, migration,

differentiation or apoptosis.

Although, fundamentally, ECM is composed of water, proteins and polysaccharides, every tissue has ECM with a unique composition and topology that is generated during tissue development through a dynamic and reciprocal biochemical and biophysical dialogue between the various cellular components and the evolving cellular and protein microenvironment. The ECM components can be divided into three major groups of molecules: insoluble (such as collagen, laminin, elastin, fibro-nectin), soluble (e.g. growth factors, chemokines, cytokines) and surface proteins of neighboring cells (cadherins). However, the composition and amount of all matrix molecules depends on cell type and location [2]. The selection of suitable extracellular matrix components may have a significant influence on in vitro cell growth. Moreover, appropriately selected ECM molecules often allow cell culturing in serum-free medium and/or without growth factors [4]. Such approach can minimize the risk of differentiation under in vitro conditions.

To date, both biological and synthetic materials have been used as ECM for in vitro cultures. However, materials derived from natural sources (e.g. collagen, laminin, fibronectin) appear to be preferable due to the presence of cell surface receptors that recognize these molecules [1].

The aim of this study was to assess the influence of different ECM components on the in vitro adhesion and proliferation of stem cells isolated from bone marrow, adipose tissue and hair follicles.

2. MATERIALS AND METHODS

The Local Bioethical Commitee of Nicolaus Copernicus University approved all procedures. In all studies, male Wistar rats (n=10) were used.

2.1. Isolation and culturing of bone marrow mesenchymal stem cells

Isolation of bone marrow was conducted using the Lennon and Caplan method [5]. Briefly, isolated rat femurs were washed with PBS supple-mented with penicillin/streptomycin (100 μg/ml) and amphotericin B (5 μg/ml) (PAA, Austria). Distal parts of the femurs were cut off and bone marrow was flushed out using DMEM/Ham's F12 supplemented with 1% antibiotics solution. Subsesquently, the bone marrow was washed twice with PBS and centrifuged at 350 x g for 10 min. Isolated cells were cultured in the above medium containing additionally 10% FBS (PAA, Austria), 10 ng bFGF (Sigma, Germany) and L-glutamine (PAA, Austria).

2.2. Isolation and culturing of adipose mesen-chymal stem cells

Adipose tissue was washed in PBS with antibiotics: penicillin/streptomycin (100 μg/ml) and amphotericin B (5 μg/ml). Subsequently, the tissue

was purified from blood vessels and incubated in collagenase type I solution (1 ml/g of tissue)

(Sigma, Germany) for 30 min in 37°C with shaking every 5 minutes. The digestion process was inhi-bited by adding an equal volume of culture medium. After that, the tissue was filtrated using a 100 µm cell strainer (BD Bioscience, USA). Thus obtained filtrate was centrifuged at 350 x g for 10 min and the cell pellet was washed twice with the culture medium. The cells were cultured in DMEM/Ham’s F12 supplemented with 10% FBS (PAA, Austria), 10 ng bFGF (Sigma, Germany), amphotericin B (5 µg/ml), penicillin/streptomycin (100 µg/ml) and L-glutamine (PAA, Austria).

2.3. Isolation and culturing of follicle stem cells

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Hair follicles were isolated using micro-tweezers and the bulge regions were cut. Thus obtained hair follicle fragments were incubated in a solution of collagenase type P (1 mg/ml) (Roche, Switzerland) and dispase (1 mg/ml) for 0.5 h at 37ºC, followed by 0.05% trypsin solution (Biomed, Poland) for additional 1.5 h. After the incubation period, the solution was centrifuged (350 x g for 10 min). Cell culture was set up on a feeder layer (3T3 cell line) in Keratinocyte Serum-Free Medium (KSFM) (Lonza, Switzerland) supplemented with peni-cillin/streptomycin (100 μg/ml) and amphotericin B (5 μg/ml).

2.4. Phenotype analysis of isolated cells

Isolated stem cells were analyzed for the presence of specific surface markers by flow cytometry. Bone marrow stem cells were charac-terized with the use of CD90 and CD34 marker, adipose mesenchymal stem cells with the use of

CD90, CD44, CD34 and CD45, while follicle stem cells with the use of cytokeratins 7, CD34 and p63. All analysis were performed according to the protocols previously described [6-8].

2.5. Evaluation of the influence of extracellular matrix proteins on the growth of stem cells

Stem cells isolated from three sources were cultured on 6-well plates coated with different ECM components such as: fibronectin, poly-D-lysine, laminin and collagen IV (BD Bioscience, USA). The number of seeded cells was 5 x 104/per well. Cells seeded on the polystyrene 6-well plate, not coated with any of the extracellular matrix components, served as a control. The cultures were run in media suitable for each type of stem cells at 37°C and 5% CO2. Every 2-3 days, the medium was changed. The cells were incubated in these conditions for 7 days. Cell viability was analyzed using the MTT assay.

Figure 1. Stem cells isolated from bone marrow (A), adipose tissue (B), hair follicle (C) 30 minutes after seeding on plates

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3. RESULTS

For phenotypic characterization bone marrow, mesenchymal stem cells were assessed for the expression of CD90 and CD34. Expression of CD90 was at high level, while staining for CD34 was negative [6]. Adipose mesenchymal stem cells sho-wed the high expression of CD90 and lower CD34 and CD44. The presence of CD45 was not detected [7]. Follicle stem cells expressed epithelial markers and were slighly positive for CD34 and p63 [8].

The analyzed stem cells showed significant differences in the ability of adhesion to the growth surface. The adhesion of bone marrow mesen-chymal stem cells to the growth surface coated with fibronectin and collagen IV was 90% in 30 min after seeding (Fig. 1A1 and A2). Both modified surfaces supported the formation of a regular monolayer of spindle-shaped cells. However, at the same time, in the cultures on laminin- and poly-D-lysine-coated surfaces, the adhesion of cells was only 60% (Fig. 1A3 and A4).

Adipose-derived mesenchymal stem cells demonstrated a 90% adhesion during 30 min after seeding on plates coated with fibronectin, collagen IV and poly-D-lysine (Fig. 1B1, B2 and B4). However, the adhesion of the same cells cultured at the same time on laminin-coated surface was only 45% (Fig. 1B3). Hair follicle stem cells cultured on collagen IV- and laminin-coated plates showed a 50% adhesion during 3 h after seeding on the modified surfaces (Fig. 1C2 and C3). However, the adhesion of these cells to the culture plates coated with fibronectin and poly-D-lysine at the same time was only 10% (Fig. 1C1 and C4). The rate of cell proliferation was determined by MTT assay after 7 days of culture. The proliferation of bone marrow mesenchymal stem cells was the fastest on plates coated with fibronectin and collagen IV compared to the control culture. The slowest growth of these cells was observed on laminin-coated surface (Fig. 2).

The best results regarding the proliferation of adipose-derived mesenchymal stem cells were observed on the control surface, as well as the surface coated with collagen IV. The slowest growth of these cells was observed on plates coated with laminin (Fig. 3).

The proliferation of hair follicle stem cells was the fastest on the control surface that was not coated with any of analyzed extracellular matrix components. On each modified surface, cell growth was about 4 times slower (Fig. 4).

Figure 2. Proliferation rate of bone marrow mesenchymal stem cells on surfaces coated with different components of extracellular matrix.

Figure 3. Proliferation rate of adipose-derived mesenchymal stem cells on plates coated with different components of extracellular matrix.

Figure 4. Proliferation rate of hair follicle stem cells on

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4. DISCUSSION

The success of an in vitro culture often depends on the creation of environment that mimics the in vivo conditions. To date, a lot of biomaterials have been tested to provide a matrix for the proper cell adhesion and proliferation. Many materials such alginate, collagen or fibrin have been used. These molecules should support cell-cell and cell-matrix interactions, as well as regulate cell proliferation, migration, matrix remodeling and tissue organization - similarly to the in vivo conditions. Moreover, materials that are used should be biocompatible: show low antigenicity, biodegradability, non-toxicity, and should be able to maintain stem cells in their undifferentiated phenotype and promote differentiation only after induction. Due to these features of ideal micro-carriers, all attention is directed to the natural ECM components [3].

Stem cells are located in different niches that serve as their reservoir in physiological conditions. That is why it seemed very interesting to demon-strate how stem cells derived from three different sources would respond to various extracellular matrix components. In order to investigate the influence of ECM proteins on the ability of adhesion and proliferation rate of the stem cells from bone marrow, adipose tissue and hair follicles, collagen IV, fibronectin, laminin and poly-D-lysine were used.

The cell membrane of mesenchymal stem cells (MSCs), isolated inter alia from bone marrow, amniotic fluid, skin and adipose tissue, has receptors of adhesion molecules, such as ICAM-1, VCAM-1 and subunits of integrins [9, 10]. MSCs produce ECM proteins, such as collagen type I and III, laminin, vimentin and osteonectin [11]. Therefore, interaction of these cells with the matrix proteins appears to be essential for their differentiation. Lanfer et al. observed that cells grown on standard polystyrene culture vessels lose their original organization observed in physiological conditions [12].

Our results show that none of the tested substances reduced the proliferation of bone marrow, as well as adipose-derived mesenchymal stem cells, with the exception of laminin. This demonstrates the biocompatibility of such modified

surfaces and possibility of using them for culturing these types of stem cells. In the case of stem cells isolated from rat bone marrow, the best proliferation rate was observed on plates coated with fibronectin and collagen IV, which also favored maintaining spindle-shaped, fibroblast-like cell morphology. Salasznyk et al. also showed that 80% of cells demonstrated adhesion during 30 min after seeding on a surface coated with fibronectin [13]. They also observed a slower proliferation of MSCs on culture dishes coated with laminin, which is consistent with the results obtained in this study. Similar findings were reported by Cool and Nurcombe [14], who demonstrated that human bone marrow mesen-chymal stem cells attached to culture plates coated with fibronectin grew much better than those attached to other analyzed modified surfaces. Our studies showed that a similar effect is obtained with fibronectin, which promotes cell adhesion and proliferation.

Culture plates coated with collagen IV also showed a positive effect on the adhesion and proliferation of rat adipose-derived stem cells. However, in this case, favorable results were also observed on control plates that were not modified. This is in agreement with the suggestion by van Dijk et al. that MSCs from different sources show strong affinity to plastic culture plates [15].

We obtained different results regarding hair follicle stem cells. The adhesion of these cells to culture surfaces was much slower than in other cell types. Moreover, from all analyzed matrix proteins, the adhesion of hair follicle stem cells was the fastest on the control polystyrene surface, not coated with any of analyzed matrix components. Much poorer adhesion of these cells was observed on all analyzed modified surfaces. These results do not coincide with the previous data. Studies using mouse hair follicle stem cells, as well as limbus epithelial cells, showed that collagen IV was the best substrate for the culture of those cells [16, 17]. Adams and Watt showed a low level of adhesion of epithelial cells to culture plates coated with laminin [18]. In our studies, we noticed slow adhesion and proliferation rate not only on collagen IV and fibronectin, but also on laminin. Such weak

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α2, α3, α3β1, α4, α5) on the cell surface [19, 20]. It does not seem that the exposure of cells to ECM proteins could induce them to synthesize the corresponding receptors. However, the ubiquity of laminin and collagen IV in the basement membrane of hair follicles, described by Jahoda et al., may explain quite rapid adhesion of hair follicle stem cells to these substrates [21].

Extracellular matrix is a multifunctional network of fibrous, which provides structural and biochemical support to all tissues. These proteins have been implicated in many cellular processes, such as migration, proliferation, differentiation or apoptosis [21].

Regardless of the tissue type, ECM consists of different components and growth factors. More recently, it has been shown that stem cells are able to respond to the mechanical properties of the matrix. Cells can respond to microenvironment and change ECM expression, which resulting in remodeling f the matrix [22]. Besides its obvious role in determining the architecture and mechanical properties, ECM strongly influences the different cell functions [21]. However, the structure of ECM in most tissues is not well understood.

5. CONCLUSIONS

Our results showed that, depending on the origin of stem cells, their response to ECM components is different and that stem cells derived from distinct sources present differences in adhesion and proliferation rates with reference to the ECM components used. This indicates that ECM is an important component of the cellular niche in the tissue, supplying critical biochemical and physical signals to initiate or sustain cellular functions. It is also possible that ECM is required for the reconstitution of the niche of stem cells in vitro.

ETHICAL APPROVAL

All procedures conducted in the experiments involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent: Informed consent was obtained from all individual participants included in the study.

AUTHORS’ CONTRIBUTION

AB - study design/planning, data collection/entry, data interpretation, literature analysis/search, wrote the initial draft of the manuscript. DP - data collection/entry, data interpretation. KR - literature analysis/search, data interpretation, statistical analy-sis, preparation of manuscript. The final manuscript has been read and approved by all authors.

TRANSPARENCY DECLARATION

The authors declare that they have no conflict of interest.

REFERENCES

1. Frisk T, Rydholm S, Andersson H, Stemme G, Brismar H. A concept for miniaturized 3-D cell culture using an extracellular matrix gel. Electrophoresis. 2005; 26: 4751-4758.

2. Haque MA, Nagaoka M, Hexig B, Akaike T. Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials. Sci Technol Adv Mater. 2010; 11: 1-10.

3. Choi JS, Kim BS, Kim JD, Choi YC, Lee EK, Park K, et al. In vitro expansion of human adipose-derived stem cells in a spinner culture system using human extracellular matrix powders. Cell Tissue Res. 2011; 345: 415-423.

4. Kleinman HK, Luckenbill-Edds L, Cannon FW, Sephel GC. Use of extracellular matrix components for cell culture. Anal Biochem. 1987; 166: 1-13. 5. Lennon DP, Caplan AI. Isolation of rat

marrow-derived mesenchymal stem cells. Exp Hematol. 2006; 34: 1606-1607.

6. Bajek A, Drewa T, Joachimiak R, Spoz Z, Gagat M, Bodnar M, et al. Myogenic differentiation of mesenchymal stem cells is induced by striated muscle influences in vitro. Curr Sign Trans Therapy. 2012; 7: 220-227.

(21)

European Journal of Biological Research 2017; 7 (3): 165-171

8. Drewa T, Joachimiak R, Bajek A, Gagat M, Grzanka A, Bodnar M, et al. Hair follicle stem cells can be driven into a urothelial‐like phenotype: an experimental study. Int J Urol. 2013; 20; 537-542. 9. Brooke G, Tong H, Levesque JP, Atkinson K.

Molecular trafficking mechanisms of multipotent mesenchymal stem cells derived from human bone marrow and placenta. Stem Cells Dev. 2008; 17: 929-940.

10. Mariotti E, Mirabelli P, Abate G, Schiattarella M, Martinelli P, Fortunato G, et al. Comparative characteristic of mesenchymal stem cells from human bone marrow and placenta: CD10, CD49d, and CD56 make a difference. Stem Cells Dev. 2008; 17: 1039-1042.

11. Lai Y, Sun Y, Skinner CM, Son EL, Lu Z, Tuan RS, et al. Reconstitution of marrow-derived extracellular matrix ex vivo: a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. Stem Cells Dev. 2010; 19: 1095-1107.

12. Lanfer B, Seib FP, Freudenberg U, Stamov D, Bley T, Bornhäuser M, Werner C. The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices. Biomaterials. 2009; 30: 5950-5958.

13. Salasznyk R, Williams W, Boskey A, Batorsky A, Plopper G. Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. J Bio Biotechnol. 2004; 1: 24-34.

14. Cool SM, Nurcombe V. Substrate induction of osteogenesis from marrow-derived mesenchymal precursors. Stem Cells Dev. 2005; 14: 632-642. 15. van Dijk A, Niessen HWM, Ursem W, Twisk JWR,

Visser FC, van Milligen FJ. Accumulation of fibronectin in the heart after myocardial infarction: a

putative stimulator of adhesion and proliferation of adipose-derived stem cells. Cell Tissue Res. 2008; 332: 289-298.

16. Li DQ, Chen Z, Song XJ, de Paiva CS, Kim HS, Pflugfelder SC. Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness. Exp Eye Res. 2005; 80: 581-590. 17. Blazejewska EA, Schlötzer-Schrehardt U, Zenkel

M, Bachmann B, Chankiewitz E, Jacobi C, Kruse FE. Corneal limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial-like cells. Stem Cells. 2009; 27: 642-652.

18. Adams JC, Watt FM. Expression of β1, β3, β4 and

β5 integrins by human epidermal keratinocytesand non-differentiating keratinocytes. J Cell Biol. 1991; 115: 829-841.

19. Jahoda CA, Mauger A, Bard S, Sengel P. Changes in fibronectin, laminin and type IV collagen distribution relate to basement membrane restructuring during the rat vibrissa follicle hair growth cycle. J Anal. 1992; 181: 47-60.

20. Gogali A, Charalabopoulos K, Constantopoulos S. Integrin receptors in primary lung cancer. Exp Oncol. 2004; 26: 106-110.

21. Chen XD. Extracellular matrix provides an optimal niche for the maintenance and ropagation of mesenchymal stem cells. Birth Defects Res C Embryo Today. 2010; 90: 45-54.

Figure

Figure 1. The inoculated flask containing the submerged fermentation medium of mandarin peel wastes

Figure 1.

The inoculated flask containing the submerged fermentation medium of mandarin peel wastes. View in document p.6
Figure 1. Stem cells isolated from bone marrow (A), adipose tissue (B), hair follicle (C) 30 minutes after seeding on plates coated with fibronectin (1), collagen IV (2), laminin (3) and poly-D-lysine (4)

Figure 1.

Stem cells isolated from bone marrow A adipose tissue B hair follicle C 30 minutes after seeding on plates coated with fibronectin 1 collagen IV 2 laminin 3 and poly D lysine 4 . View in document p.17
Figure 1. Serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities of experimental rat groups

Figure 1.

Serum aspartate aminotransferase alanine aminotransferase and alkaline phosphatase activities of experimental rat groups. View in document p.28
Figure 3. Serum total protein and albumin concentrations of experimental rat groups.

Figure 3.

Serum total protein and albumin concentrations of experimental rat groups . View in document p.29
Figure 2. Serum total bilirubin concentration of experimental rat groups.

Figure 2.

Serum total bilirubin concentration of experimental rat groups . View in document p.29
Figure 5. Serum lipid profile of experimental rat groups.

Figure 5.

Serum lipid profile of experimental rat groups . View in document p.30
Figure 4. Serum creatinine and urea concentrations of experimental rat groups.

Figure 4.

Serum creatinine and urea concentrations of experimental rat groups . View in document p.30
Figure 6. Photomicrograph sections of hepatic tissues (H&E x 400).  Group 1: Normal histologyshowing normal central vein (blue arrow)

Figure 6.

Photomicrograph sections of hepatic tissues H E x 400 Group 1 Normal histologyshowing normal central vein blue arrow . View in document p.31
Figure 1. Different stages of in-vitro regeneration of A. saccata from nodal explant. A = plant in wild condition,                 B = inoculation of nodal explants in MS medium, C-D = initial days after inoculation in MS medium, E-H = direct organogenesis

Figure 1.

Different stages of in vitro regeneration of A saccata from nodal explant A plant in wild condition B inoculation of nodal explants in MS medium C D initial days after inoculation in MS medium E H direct organogenesis. View in document p.44
Table 2. Effect of cytokinins and auxins individually and in combinations for organogenesis from nodal explants of                    A

Table 2.

Effect of cytokinins and auxins individually and in combinations for organogenesis from nodal explants of A. View in document p.45
Table 1. Effect of cytokinins and auxins individually and in combinations for organogenesis from nodal explants              of A

Table 1.

Effect of cytokinins and auxins individually and in combinations for organogenesis from nodal explants of A. View in document p.45
Figure 2. Different stages of in-vitro regeneration of A. cathcartii from nodal explant

Figure 2.

Different stages of in vitro regeneration of A cathcartii from nodal explant. View in document p.46
Table 1. Clinical data and demographic characteristics of patients (n=205).

Table 1.

Clinical data and demographic characteristics of patients n 205 . View in document p.54
Table 2.dipstick test altered among clinical data.
Table 2 dipstick test altered among clinical data . View in document p.54
Table 1. List of species accepted to-date (ordered alphabetically).

Table 1.

List of species accepted to date ordered alphabetically . View in document p.59
Figure 1. Chemical structure of OTA.

Figure 1.

Chemical structure of OTA . View in document p.59
Table 2. Black aspergilli in agricultural commodities.

Table 2.

Black aspergilli in agricultural commodities . View in document p.62
Table 4. Ochratoxins produced by black aspergilli isolated from agricultural commodities

Table 4.

Ochratoxins produced by black aspergilli isolated from agricultural commodities. View in document p.64
Table 7. Plant diseases caused by Aspergillus niger.

Table 7.

Plant diseases caused by Aspergillus niger . View in document p.65
Table 6. Fumonisins produced by black aspergilli isolated from agricultural commodities

Table 6.

Fumonisins produced by black aspergilli isolated from agricultural commodities. View in document p.65
Figure 1. Structure of important chemical constituent of

Figure 1.

Structure of important chemical constituent of . View in document p.75
Table 1. Antibacterial activities of the ethanolic extracts of 26 endophytic fungi against three different strains of each of Gram (+) and Gram (-) bacteria

Table 1.

Antibacterial activities of the ethanolic extracts of 26 endophytic fungi against three different strains of each of Gram and Gram bacteria . View in document p.88
Table 2. Antifungal activities of the ethanolic extracts of 26 endophytic fungi against some strains of yeasts, dermatophytic and keratinophilic fungi

Table 2.

Antifungal activities of the ethanolic extracts of 26 endophytic fungi against some strains of yeasts dermatophytic and keratinophilic fungi . View in document p.89
Table 3. Anti-inflammatory activity of ethanolic extracts of selected endophytic strains

Table 3.

Anti inflammatory activity of ethanolic extracts of selected endophytic strains . View in document p.91
Figure 1. Structure of chrysin.

Figure 1.

Structure of chrysin . View in document p.96
Table 1. The type of active substances and the cell line on which they act.

Table 1.

The type of active substances and the cell line on which they act . View in document p.100
Figure 1. Phosphorus cycle in soil.

Figure 1.

Phosphorus cycle in soil . View in document p.107
Table 1. Forms of phosphorus extracted by Hedley et al. [22].

Table 1.

Forms of phosphorus extracted by Hedley et al 22 . View in document p.107
Table 2. Literature reports on soil P fractions (mg kg-1) in surface soil (0-15 cm) under different vegetation types

Table 2.

Literature reports on soil P fractions mg kg 1 in surface soil 0 15 cm under different vegetation types. View in document p.110
Figure 2.  Release of P through the action of low molecular weight organic acids and other naturally occurring chelates

Figure 2.

Release of P through the action of low molecular weight organic acids and other naturally occurring chelates. View in document p.113

References

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